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首页> 外文期刊>Biochemistry >Dimerization is crucial for the function of the Na+/H+ exchanger NHE1
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Dimerization is crucial for the function of the Na+/H+ exchanger NHE1

机译：二聚化对于Na + / H +交换器NHE1的功能至关重要

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摘要

The Na+/H+ exchanger 1 (NHE1) exists as a homo-dimer in the plasma membranes. In the present study, we have investigated the functional significance of the dimerization, using two nonfunctional NHE1 mutants, surface-expression-deficient G309V and transport-deficient E262I. Biochemical and immunocytochemical experiments revealed that these NHE1 mutants are capable of interacting with the wild-type NHE1 and, thus, forming a heterodimer. Expression of G309V retained the wild-type NHE1 to the ER membranes, suggesting that NHE1 would first form a dimer in the ER. On the other hand, expression of E262I markedly reduced the exchange activity of the wild-type NHE1 through an acidic shift in the intracellular pH (pH(i)) dependence, suggesting that dimerization is required for exchange activity in the physiological pH(i) range. However, a dominant-negative effect of E262I was not detected when exchange activity was measured at acidic pH(i), implying that one active subunit is sufficient to catalyze ion transport when the intracellular H+ concentration is sufficiently high. Furthermore, intermolecular cysteine cross-linking at extracellular position Ser(375) with a bifunctional sulfhydryl reagent dramatically inhibited exchange activity mainly by inducing the acidic shift of pH(i) dependence and abolished extracellular stimuli-induced activation of NHE1 without causing a large change in the affinities for extracellular Na+ or an inhibitor EIPA. Because monofunctional sulfhydryl regents had no effect, it is likely that cross-linking inhibited the activity of NHE1 by restricting a coupled motion between the two subunits during transport. Taken together, these data support the view that dimerization of two active subunits are required for NHE1 to possess the exchange activity in the neutral pH(i) range, although each subunit is capable of catalyzing transport in the acidic pH(i) range.
机译：Na + / H +交换剂1（NHE1）以均二聚体的形式存在于质膜中。在本研究中，我们研究了使用两个非功能性NHE1突变体，表面表达缺陷型G309V和运输缺陷型E262I的二聚化的功能意义。生化和免疫细胞化学实验表明，这些NHE1突变体能够与野生型NHE1相互作用，从而形成异二聚体。 G309V的表达将野生型NHE1保留在ER膜上，表明NHE1将首先在ER中形成二聚体。另一方面，E262I的表达通过细胞内pH（pH（i））依赖性的酸性变化而明显降低了野生型NHE1的交换活性，这表明在生理pH（i）中交换活性需要二聚化范围。但是，当在酸性pH（i）下测量交换活性时，未检测到E262I的显性负作用，这意味着当细胞内H +浓度足够高时，一个活性亚基足以催化离子转运。此外，分子间半胱氨酸在细胞外位置Ser（375）与双功能巯基试剂的交联显着抑制交换活性，主要是通过诱导pH（i）依赖性的酸移并取消细胞外刺激诱导的NHE1活化，而不会引起NHE1的大变化。对细胞外Na +或抑制剂EIPA的亲和力。因为单官能巯基试剂没有作用，所以交联很可能通过限制运输过程中两个亚基之间的耦合运动而抑制了NHE1的活性。综上所述，这些数据支持这样的观点，即NHE1在中性pH（i）范围内具有交换活性需要两个活性亚基的二聚化，尽管每个亚基都能够催化酸性pH（i）范围内的转运。

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